The present invention relates generally to cell transfection, and, more particularly, to an apparatus and method of electroporation that prevents and/or reduces arcing across a cuvette containing a sample of biological cells.
Electroporation is a process by which high-voltage (typically high energy) electric potentials are used to create temporary holes ("pores") in the walls of biological cells. These pores allow the passage of large molecules (e.g., DNA) into the cell, before the cell eventually closes the pores. As a consequence, electroporation can be used to program a cell to produce proteins specified by the DNA (bacterial cells for example can be caused to produce human insulin). Electroporation is, therefore, an extremely powerful tool, since a 5-msec application of a high-energy pulse can create openings in millions of cells at the same time. Cells that have received DNA in this manner can then be grown in a nutritive broth to produce an aggregate, which generates large quantities of some desirable complex molecule. Typically, a biological cell can produce compounds in seconds or minutes whereas a conventional synthesis may require a week or more when performed in a laboratory using chemical synthesis procedures.
Previous patents (U.S. Pat. Nos. 4,750,100, 5,656,926 and 5,642,035), each of which is incorporated herein by reference, describe the use of semiconductor-controlled rectifiers (SCRs) in a stacked-cell arrangement to produce a solid-state high-voltage (HV) switch capable of controlling 3000V pulses at currents of 1500 amperes (or higher, depending on the SCR). Increasing the number of SCR cells allows the control of higher voltage pulses. The general method of producing such pulses is shown in FIG. 1 attached hereto.
In FIG. 1, a charging means 4, consisting of a linear or switcher current source, is controlled by a microcontroller 10 through an isolated control line 18. The microcontroller 10 turns on the charging means 4, thereby causing charge to accumulate in an HV capacitor 6. As the voltage across the HV capacitor 6 rises, the microcontroller 10 monitors the increasing voltage, using a voltage divider 12 consisting of two or more resistors. When the voltage has reached a predetermined value, either by embedded program or external setting by an operator, the microcontroller 10 triggers the HV switch 8 (consisting of the SCR cells) through a trigger circuit 14. The HV switch 8 effectively connects the HV capacitor 6 to the output of the system, which in turn connects to a cuvette 16 or other sample holder containing cells and DNA (or other) compounds. The resistance of the sample in the cuvette 16 may be effectively 10 ohms at high voltage. Hence, without a current limiting resistor, large currents could flow and destroy the HV switch 8. Later embodiments, such as those in U.S. Pat. Nos. 5,656,926 and 5,642,035, for example, use a 1.5-ohm series resistor with feedback through the microcontroller to compensate for voltage drops across the resistor.
Another concern in the use of such a system is arc-over. If the voltage at the cuvette 16 is too high, an arc-over may occur. Arc-overs lower the cuvette's 16 effective resistance to 1.0 ohms or less. Hence, at 3000V (with 1.5 ohms current limiting resistance), 2000 amperes may flow. Typical SCRs used for such an application will tolerate such currents. However, the arc will cause a visible and auditory event at the cuvette 16, which may expel the sample, destroy cells, and startle the operator.
In the electroporator system of U.S. Pat. No. 4,750,100, no specific current limiting resistor is incorporated, since such a resistor would cause a large voltage drop. (The resistance required by the resistor would have to be 1.5 ohms, which is significant compared to that of the sample, whose resistance is 10 ohms.) U.S. Pat. No. 4,750,100 does, however, describe the use of the SCR cells to produce an appropriate HV switch. In U.S. Pat. Nos. 5,656,926 and 5,642,035 a system that measures sample resistance is included. This allows the microprocessor to compensate for the voltage drop across a current limiting resistor by charging the HV capacitor to a somewhat higher voltage. Other novel portions of the design are described in the same two patents.
Lack of arc protection in the electroporator system of U.S. Pat. No. 4,750,100 was acceptable since arcs were infrequent, until the system was later used on bacterial cells. Bacteria require the use of higher voltages, thereby increasing the likelihood of arcs. Bacteria electroporation in those systems requires a special box containing the current limiting resistor (which might be forgotten by the user to the detriment of the instrument) or the incorporated arc protection of the instruments disclosed in U.S. Pat. Nos. 5,656,926 and 5,642,035.
A better system would be one that, not only provides protection from arcs, but one that limits the time in which an arc is applied to a sample. Such a system would have less of a chance of startling users and would prevent destruction of valuable cells. The present invention provides a solution that addresses these issues.